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Activity 2-1 - Plasmids Isolation

Last updated

Aug 3, 2025
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- Activity 2-0 - Introduction to Plasmid and Enzyme p450
- Activity 2-2 - DNA Analysis via Restriction Enzymes

Victor Pham
Irvine Valley College

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Learning Objectives

Define what a plasmid is and explain why it is biologically and biotechnologically important.
Describe how plasmids can carry genes like antibiotic resistance or metabolic enzymes (e.g., P450).
Explain how plasmids are introduced into bacteria and how they replicate.
Outline the key steps and principles behind the plasmid mini prep procedure.
Understand the purpose and function of buffers P1, P2, P3, binding buffers, and wash/elution steps.

Definition: Term

Plasmid: A small, circular, double-stranded DNA molecule found in bacteria, independent of chromosomal DNA.
Supercoiling: A compact DNA structure resulting from the twisting of the DNA helix, aiding plasmid stability and extraction.
Vector: A vehicle used to deliver genetic material into cells (e.g., plasmids).
Transformation: The process of introducing foreign DNA (like a plasmid) into a bacterial cell.
Miniprep: A laboratory technique used to isolate plasmid DNA from bacterial cells.
Alkaline lysis: A method using high pH to break open cells and selectively isolate plasmid DNA.
EDTA: A molecule that chelates metal ions, protecting DNA from degradation by nucleases.
SDS: A detergent that lyses cells and denatures proteins.
Potassium acetate: Neutralizes the lysis solution and causes precipitation of chromosomal DNA and proteins.
Spin column: A silica-based tool that binds DNA in high-salt conditions for purification.

Background Questions

Why would a researcher choose to use a plasmid instead of integrating a gene directly into the bacterial chromosome?
What makes plasmid DNA easier to isolate than genomic DNA?
Why is it important to include RNase and EDTA in the resuspension buffer?

Note

Plasmids are powerful tools in genetic engineering, especially for cloning and expression studies.
Each buffer in the miniprep has a distinct role in protecting, releasing, purifying, or binding DNA.
Understanding the chemical basis of lysis and DNA precipitation helps troubleshoot common lab issues (e.g., low DNA yield or contamination).
Hands-on practice with miniprep connects molecular theory with real-world biotechnology workflows.

What is a Plasmid and Why It Matters

A plasmid is a small, circular, double-stranded DNA molecule distinct from the chromosomal DNA found in bacteria. These plasmids often carry genes that provide a survival advantage, such as antibiotic resistance or metal ion resistance. For example, a plasmid carrying a gene for ampicillin resistance allows bacteria to survive in media containing this antibiotic. Plasmids are supercoiled, making them more compact, more stable, and easier to isolate than chromosomal DNA. In molecular biology, plasmids are used as vectors—vehicles to transfer genetic material. If a researcher wants to study the function of the cytochrome P450 gene, they can clone this gene into a plasmid, transform it into E. coli, and then harvest that plasmid from the bacteria after amplification.

After inserting the P450 gene into a plasmid, the next step is transformation—introducing the recombinant plasmid into competent E. coli cells. These bacteria, once transformed, replicate the plasmid along with their own DNA during cell division. This natural amplification ensures that from a single transformed colony, you can culture millions of bacteria, each carrying multiple copies of the plasmid. This is essential because it provides a mass production system for the DNA you want to study or manipulate.

Principle of Plasmid Mini Prep

To analyze the plasmid DNA (e.g., verify the presence of the P450 gene by restriction digestion), we need to extract it from the bacterial cells. The Plasmid Mini Prep is a quick and efficient method to purify plasmid DNA, separating it from chromosomal DNA, proteins, RNA, and other cellular components. This method relies on alkaline lysis, developed by Birnboim and Doly in 1979, which takes advantage of structural differences between plasmid and genomic DNA to selectively isolate plasmids.

1. Resuspension in Buffer P1

Bacterial cells from a liquid culture are first harvested by centrifugation. The pellet is then resuspended in Buffer P1, which contains:

EDTA: A chelating agent that binds divalent metal ions (like Mg²⁺ and Ca²⁺). These ions are essential cofactors for nucleases like DNase, which could degrade DNA. By removing metal ions, EDTA inhibits DNases, protecting the plasmid DNA. If EDTA were omitted, any endogenous DNases could become active during cell lysis and degrade your plasmid DNA, leading to poor yields or fragmented plasmids.
RNAse A: An enzyme that degrades RNA. This ensures that the final preparation is free from RNA contamination, allowing you to work exclusively with DNA.

2. Lysis with Buffer P2

After resuspension, Buffer P2 is added. This step causes the cell to burst open, releasing its contents. Both DNA types become single-stranded, and cellular proteins and lipids start to precipitate or denature. This solution contains:

Sodium dodecyl sulfate (SDS): An anionic detergent that disrupts the cell membrane and denatures proteins. SDS disrupts the lipid bilayer of the bacterial membrane, much like soap breaks down grease.
NaOH (alkaline): Raises the pH drastically, causing denaturation of chromosomal and plasmid DNA. The high pH simultaneously denatures the bacterial proteins and unwinds all DNA strands.

3. Neutralization with Buffer P3

To stop the harsh alkaline reaction, Buffer P3 is added. At this point, a critical physical property difference is exploited: plasmid DNA is small and supercoiled, so it can quickly reanneal and stay in solution. In contrast, chromosomal DNA is large and entangled, so it aggregates and precipitates. It contains:

Potassium acetate: It neutralizes the pH, allowing DNA strands to renature. The potassium ions also form an insoluble complex with SDS and denatured proteins, which precipitate out of solution. Think of plasmid DNA like a rubber band—it snaps back into shape when tension is released. Genomic DNA is like a long rope—it tangles up and settles out.

4. Centrifugation and DNA Binding

The resulting mixture is centrifuged at high speed. The supernatant is then passed through a silica spin column. Under high-salt conditions, DNA binds to silica. Other contaminants pass through during the wash steps.

The pellet contains cell debris, precipitated proteins, and genomic DNA.
The supernatant contains your clean plasmid DNA. DNA sticks to silica under certain salt conditions much like Velcro attaches when pressed together. Later, it can be eluted with a low-salt or water-based buffer.

5. Elution

Finally, the bound plasmid DNA is eluted with a low-salt buffer or sterile water. This DNA can now be used for downstream applications like restriction digestion, sequencing, PCR, or transformation into another organism. For example, if your plasmid carries the P450 gene, a restriction digest can be used to confirm its presence. Specific enzymes will cut the DNA at known sites, generating fragments of predictable sizes when run on an agarose gel. The pattern will tell you whether your plasmid carries the gene and whether it's in the right orientation.

Image of a flow chart summarizing Plasmid Mini Prep. Image created by Dr. Victor Pham's student, Geneva Anh Thy Doan.

Plasmid Mini Prep Protocol (ZymoPURE Kit)

Goal: Isolate plasmid DNA from E. coli cultures containing either pET3 or pT7BM3 plasmids.

Materials Needed:

Disinfectant spray
70% Ethanol (Flinn Scientific #E0013)
Biohazard tip waste and liquid waste containers
Microcentrifuge (Carolina #214061)
1.5 mL microcentrifuge tubes and tube rack
E. coli culture with pET3 plasmid
E. coli culture with pT7BM3 plasmid
ZymoPURE Plasmid Miniprep Kit (Zymo #D4211)
Buffer P1 (keep refrigerated at 4°C)
Micropipettes: P10, P20, P200, P1000 + sterile tips

Safety note: Disinfect your workspace and wash your hands before and after the procedure. Use proper pipetting technique and wear gloves throughout.

🧬 Procedure

Each team member should handle one culture type:
- Group A: pET3 plasmid
- Group B: pT7BM3 plasmid
Use a P1000 pipette to transfer 1.5 mL of bacterial culture into a labeled 1.5 mL microcentrifuge tube.
- Label clearly with group name and plasmid type.
Centrifuge the tube at maximum speed (≥10,000 rpm) for 1 minute.
Carefully pour off or pipette out the supernatant without disturbing the white pellet at the bottom.
Repeat steps 2–4 three more times, using the same microcentrifuge tube each time.
- This concentrates your bacteria into one pellet for better plasmid yield.
Add 250 μL of Buffer P1 using a new tip.
Pipette up and down or vortex gently until the pellet is fully dissolved—no clumps should remain.
Add 250 μL of Buffer P2 to the tube.
Gently invert the tube 6 times (do NOT vortex). The solution should turn viscous and purple.
Let the tube sit for 3 minutes (no more than 5 minutes).
Add 250 μL of Buffer P3 and immediately invert 6 times. The solution will become cloudy and yellow as cell debris and proteins precipitate.
Spin the tube at maximum speed for 5 minutes.
You will see a white pellet at the bottom—this contains cellular debris and chromosomal DNA.
Using a P1000 pipette, carefully transfer all of the clear supernatant into a new labeled 1.5 mL tube, avoiding the white pellet.
- ️Discard the tube with the pellet.
Add 260 μL of Binding Buffer to the new tube.
Invert gently for 15 seconds.
Pour the mixture into the Zymo spin column (attached to a collection tube).
Let it sit for 1 minute, then centrifuge for 1 minute.
- Discard the flow-through from the collection tube.
- Save the Spin Column, which contains your plasmids in the silica beads.
- Reattach the collection tube to the spin column
Add 800 μL of Wash 1, centrifuge for 1 minute, and discard flow-through.
Add 800 μL of Wash 2, centrifuge for 1 minute, and discard flow-through.
Add 200 μL of Wash 2 again, centrifuge for 1 minute, and discard flow-through.
Centrifuge the spin column one more time for 1 minute to remove any leftover ethanol from the wash buffers.
- Residual ethanol can inhibit downstream reactions like PCR or restriction digest.
- (If there's still liquid in the spin column, continue spinning it down until there's no more liquid remaining)
Move the spin column into a new, labeled 1.5 mL microcentrifuge tube.
Snap off the cap of the tube.
Add 25 μL of Elution Buffer directly to the white silica membrane of the spin column.
Let it sit for 1 minute, then centrifuge for 2 minutes at maximum speed.
Cap and label the tube as either “Plasmid – pT7BM3” or “Plasmid – pET3a”.
Place the tube in the freezer for storage.
- Discard the used Silica spin column.
Measure your DNA via nanodrop

Parameter	Ideal Range	Notes
Concentration	>0.1 µg/µL	This also depends on how much bacteria was used from step 1
A260/A280	>1.8	Protein/phenol contamination indicator
A260/A230	>2.0	Salt/guanidine/alcohol contamination indicator

Summary

Plasmids are essential tools for gene cloning, protein production, and synthetic biology.
Transformation allows bacteria to amplify plasmids, creating a renewable source of genetic material.
The mini prep process leverages molecular biology principles (like DNA binding to silica in salt) to purify plasmid DNA.
Proper buffer usage and careful pipetting are critical for successful DNA extraction.

Post-Lab Questions

What step in the miniprep protocol is most likely to affect your DNA yield if not done carefully? Why?
How does each buffer in the mini prep process help isolate plasmid DNA? Which buffer surprised you the most in terms of function?
Suppose you wanted to check if your plasmid contains the correct gene. What would be your next steps after the miniprep?
How does plasmid-based antibiotic resistance affect modern medicine and public health?
What would happen if you forgot to add Buffer P3?
Why is it important to remove all ethanol from the spin column before elution?
Why are plasmids useful for studying genes like cytochrome P450?